Guanidine derivatives, methods of preparing them and their use as drugs

ABSTRACT

The invention concerns new guanidine derivatives of formula (I), methods of preparing them and their use in drugs containing compounds, of formula (I), wherein                    
     wherein x is —NH—NH—CH 2 R 1  and R 1  denotes C 8  to C 20  alkyl.

This is the national phase of PCT/EP97/02658 filed May 23, 1997, now WO97/45401.

The present invention relates to new guanidine derivatives, processesfor preparing them and their use as sphingomyelinase inhibitors andpharmaceutical compositions which contain these compounds. The guanidinederivatives of the present invention correspond to the general formulaI:

wherein

X denotes R₁, —NHR₁, —NH—NH—CHR₁R₂, —NH—N═CR₁R₂,

R₁ and R₂ independently of each other denote hydrogen, astraight-chained or branched C₃₋₂₀-alkyl, C₃₋₂₀-cycloalkyl group, anadamantyl, norbornyl, tricyclodecyl, benzyl, furyl, pyridyl, indolyl,quinolyl, anthracenyl, phenanthryl, perinaphthyl or quinuclidinyl group,wherein the above-mentioned straight-chained or branched C₃₋₂₀-alkylgroup may be substituted by a hydroxy or C₁₋₄-alkoxy group, a halogenatom or an amino group and the above-mentioned C₃₋₂₀-cycloalkyl groupmay be substituted by a hydroxy, C₁₋₄-alkoxy, C₁₋₄ -alkyl group or by ahalogen atom or an amino group, and wherein, if X denotes —NH—N═CR₁R₂,only one of the substituents R₁ and R₂ may represent hydrogen

optionally in the form of individual optical isomers, mixtures of theindividual isomers or racemates, tautomers or geometrical isomers e.g.cis/trans-isomers as well as in the form of the free bases or thecorresponding acid addition salts with pharmacologically acceptableacids.

Preferred compounds of general formula I are those wherein

X denotes —NH—NH—CH₂R₁ and —NH—N═CHR₁

R₁ denotes C₈₋₂₀-alkyl (branched or unbranched).

Particularly preferred compounds of general formula I are those wherein

X denotes —NH—NH—CH₂R₁ and —NH—N═CHR₁ and

R₁ denotes an unbranched decyl group.

The compounds according to the invention have valuable pharmacodynamicand biochemical properties and can therefore be used advantageously inresearch and in human and veterinary medicine.

Surprisingly it has been found that the aminoguanidines and amidinesaccording to the invention have beneficial sphingomyelinase-inhibiting,antimicrobial, antiviral, anti-inflammatory (e.g. anti-shock) activitiesand effects on cell growth.

The compounds according to the invention are prepared by reacting analdehyde or ketone of formula R₁CHO or R₁COR₂ with aminoguanidine. Thereaction is usually carried out in an inert organic solvent, e.g. achlorinated hydrocarbon, such as dichloromethane or chloroform, or anaromatic hydrocarbon such as benzene or toluene. The reaction ispreferably carried out by removing the water formed from theequilibrium, e.g. using a water separator. The reaction may be carriedout over a wide temperature range but is generally performed at elevatedtemperature, particularly at a temperature in the range from about 60°C. up to the boiling point of the reaction mixture. In addition, thecompounds according to the invention may be prepared by methods knownfrom the prior art.

The starting compounds are known or may be prepared by known methods.

The pharmaceutical compositions according to the invention contain oneof the above-mentioned compounds of general formula I in a conventionalsolid or liquid pharmaceutical carrier. The compounds according to theinvention may also be combined with known active substances.

The compounds according to the invention are characterised byanti-inflammatory (e.g. anti-shock), antimicrobial, antitumoral and, inparticular, antiviral effects. The antiviral spectrum of activityincludes, for example, herpes, vesicular stomatitis, HIV and papillomaviruses. It has also been found that the compounds according to theinvention influence the growth of tumour cells. They may be used totreat carcinomas, e.g. carcinoma of the large intestine, sarcomas orleukaemias.

In general, it is found that these substances according to the inventionbring about an NF-kappaB-dependent immunosuppression.

The compounds according to the invention can therefore be used to treatthe following diseases:

A. Systemic inflammatory reactions

sepsis-causing diseases

gram-positive sepsis

gram-negative sepsis

fungal sepsis

agranulocytosis (neutropenic fever)

urinary infections (urosepsis)

general infections with meningococci (meningococcaemia)

trauma/haemorrhage

burns

injuries caused by ionising radiation

acute pancreatitis

adult respiratory distress syndrome (ARDS)

B. Reperfusion syndrome

post pump syndrome

ischaemia-induced reperfusion injury

C. Cardiovascular disease:

cardiac stun syndrome

myocardial infarction

congestive heart failure

arteriosclerosis

D. Infectious diseases:

papilloma virus infection

herpes virus infection

HIV infection/HIV neuropathy

meningitis

hepatitis

septic arthritis

peritonitis

pneumonia

bronchitis

epiglottitis

E. coli 0157:H7 infection

haemolytic uremic syndrome/thrombolytic thromocytopenic purpura

malaria

Dengue haemorrhagic fever

Leishmaniasis

leprosy

toxic shock syndrome

Streptococcal myositis

gas gangrene

myobacterium tuberculosis infections

myobacterium avium intracellular infections

pneumocystosis

pelvic inflammatory disease

orchitis/epidydimitis

Legionella

lyme disease

influenza A virus infection

diseases caused by Epstein-Barr virus

viral-associated haemaphagocytic syndrome

viral encephalitis/aseptic meningitis

E. Gynaecological applications

premature labour

miscarriage

infertility

F. Inflammatory diseases/autoimmune diseases:

rheumatoid arthritis/seronegative arthropathy

emphysema bronchitis (chronic obstructive pulmonary disease COPD)

osteoarthritis

inflammatory bowel disease

Crohn's disease

systemic lupus erythematosis

iridocyclitis/uveitis/optic neuritis

idiopathic pulmonary fibrosis

systemic vasculitis/Wegner's granulomatosis

sarcoidosis

orchitis/vasectomy reversal procedures

H. Allergic/atopic diseases:

asthma

allergic rhinitis

eczema

allergic contact dermatitis

allergic conjunctivitis

hypersensitive pneumonitis

I. Malignant disease:

tumour therapy in combination with chemotherapy, radiotherapy andcytokine treatment such as TNF-α treatment of sarcomas, carcinomas andleukaemias

ALL

AML

CML

CLL

breast cancer

small-cell and non-small-cell bronchial carcinoma

squamous cell carcinoma

Hodgkin's disease, non-Hodgkin's lymphoma

multiple melanoma

Kaposi's sarcoma

colorectal carcinoma

nasopharyneal carcinoma

malignant histiocytosis

paraneoplastic syndrome/hypercalcaemia of malignancy

J. Transplant complications

rejection reactions after transplant

graft versus host reactions

K. Cachexia

L. Congenital diseases:

cystic fibrosis

familial hematophagocytic lymphohistiocytosis

sickle cell anaemia

M. Skin diseases:

psoriasis

alopecia

N. Neurological diseases/chronic and acute neurodegeneration

multiple sclerosis

Parkinson's disease

Down's syndrome

stroke

skull/brain trauma

migraine

O. Diseases of the kidneys:

nephrotic syndrome

haemodialysis

uraemia

P. Various toxicities:

OKT3 therapy

anti-CD3 therapy

cytokine therapy

chemotherapy

radiation therapy

chronic salicylate intoxication

Q. Metabolic/idiopathic diseases:

Wilson's disease

haemachromatosis

alpha-1-antitrypsin deficiency

diabetes

Hashimoto's thyroiditis

osteoporosis

hypothalamic pituitary adrenal axis evaluation

primary biliary cirrhosis

In vitro investigations in plaque reduction tests using differentviruses showed an inhibition of growth at substance concentrations offrom 0.1 to 1000/μg/ml. The toxicity of the substances according to theinvention is relatively low. They may be used in particular as effectivepreventative or therapeutic agents against influenza, AIDS or herpesdiseases of the skin and mucous membranes. The daily dose for adultsduring the disease is of the order of about 5 to 1000 mg of activesubstance per day.

The compounds according to the invention may be administered byparental, subcutaneous, intravenous, intramuscular and intraperitonealroute. In this case, the carrier substance is a sterile liquid such aswater or oil, the oil being of vegetable, animal or synthetic origin.Conventional glucose solutions are used as the injectable solutions. Theliquid carriers for the injectable solutions generally contain 0.5 to26% by weight of active substance. The compounds according to theinvention may be administered orally with equal success. The compoundsare also suitable for treating pneumonia and are administered in theform of a vapour or spray to the oral and nasal cavity. For oraladministration, compositions in the form of tablets, capsules, powders,solutions, suspensions or elixirs are particularly preferred. Thequantity of active ingredient in these preparations is at least 1% byweight, based on the total weight of the composition. The activesubstances according to the invention may also be administeredtopically, e.g. in ointments, creams, emulsions or lotions.

The Examples which follow show some possible formulations for thepreparations:

FORMULATION EXAMPLES

1. Tablets

Composition:

Active substance according to the 20 parts by weight invention Stearicacid 6 parts by weight Glucose 474 parts by weight

The ingredients are processed in the usual way to form tablets weighing500 mg. If desired, the content of active substance may be increased orreduced and the quantity of glucose reduced or increased accordingly.

2. Suppositories

Composition:

Active substance according to the 100 parts by weight invention Powderedlactose 45 parts by weight Cocoa butter 1555 parts by weight

The ingredients are processed in the usual way to form suppositoriesweighing 1.7 g.

3. Powder for Inhalation

Micronised powdered active substance (compound of formula I; particlesize about 0.5 to 7 μm) is packed into hard gelatine capsules in aquantity of 5 mg, optionally with the addition of micronised lactose.The powder is inhaled from conventional inhalers, e.g. according to DE-A33 45 722, to which reference is hereby made.

The compounds according to the invention can be prepared starting fromcompounds known from the prior art, using the processes described in thefollowing Examples, inter alia. Other different embodiments of theinvention and processes will be apparent to anyone skilled in the artfrom the present specification. However, it is expressly pointed outthat these Examples and the associated specification are intended solelyfor purposes of explanation and should not be regarded as restrictingthe invention. Reference is further made to German Patent Application P196 21 038.0 for additional information.

Example 1

Preparation of 1-(undecylideneamino)guanidine [C11AG]

1 mol (170.3 g) of undecanol, 1.1 mol (150 g) of aminoguanidine hydrogencarbonate and 1 g of p-toluenesulphonic acid are mixed with 500 ml oftoluene and refluxed with stirring. As soon as 2 mol of water have beenseparated using the water separator, the mixture is allowed to cool,concentrated by rotary evaporation and the dark red oil is taken up in250 ml of petroleum ether 40/60°. The precipitate formed is suctionfiltered and washed again with petroleum ether. For recrystallisationthe precipitate is dissolved in ethyl acetate and mixed with petroleumether at boiling temperature (boiling range 40 to 60° C.) untilbeginning to turn cloudy. Fine crystals are obtained, m.p. 101° C. Thestructure and purity of the compound were confirmed by analytical andspectroscopic data.

The other compounds mentioned in Example 3 are prepared analogously.

Example 2

Preparation of 1-(undecylamino) guanidine [H₂C11AG]

1.2 g of 1-(undecylideneamino)guanidine are placed in an autoclave andhydrogenated over a period of 12 hours in the presence of 0.1 g of 10%palladium on activated charcoal as hydrogenation catalyst in 20 ml of100% acetic acid under a hydrogen pressure of 60 bar at ambienttemperature. Then the catalyst is filtered off and the colourlesssolution is evaporated to dryness in vacuo.

In this way the title compound is isolated, after recrystallisation fromethyl acetate, in the form of colourless crystals melting in the rangefrom 70-72° C. in quantitative yield.

Example 3

The virostatic properties were determined by in vitro tests. Thefollowing virus strains were used:

herpes virus

vesicular stomatitis virus

BVI 1

Cell cultures (monkey kidney cells or human fibroblasts) are infectedwith herpes and a series of cultures are exposed to medium containingvarious concentrations of the test substance. After 24 hours theconcentration of the virus descendants in the cell culture supernatantis determined by plaque assays. The concentration of substance at whichthe virus replication is inhibited by 50% (IC₅₀) is determined fromdosage/activity curves.

The results obtained from some substances by way of example are listedin the following Table.

Substance IC₅₀ μM 1 - (octylidene-amino) guanidine 49.7 1 -(nonylidene-amino) guanidine 29.0 1 - (decylidene-amino) guanidine 28.91 - (undecylidene-amino) guanidine 6.8 1 - (dodecylideneamino) guanidine3.2 1 - (anthracen-9-ylmethylene-amino) guanidine 1.5 1 -(indol-3-ylmethylene-amino) guanidine 19.8 1 -(phenalen-1-ylidene-amino) guanidine 45.4

Example 4

Protection from endotoxic shock by C11AG is illustrated by FIG. 1:

Mice (strain NMRI/Nu, 8 weeks old, female) were each given 0.2 mg ofendotoxin from E. coli (Sigma, Munich) by intraperitoneal route. The 10control animals, who had been given 0.2 ml of 5% glucose subcutaneously,died within 24 hours. Nine animals were injected subcutaneously with 50mg/kg of C11AG 30 minutes before the endotoxin treatment. Of this group,only 2 animals died.

Example 5

Inhibition of Collagen-induced Arthritis in the Mouse

An autoimmune reaction against cartilagenous tissue was produced byinjecting collagen into DBA/I mice as described (Holmdahl, R. et al.,Immunology, 65, 305-310, 1988). Groups of 10 animals were used ascontrol or were given 50 mg/kg or 100 mg/kg of C11AG per day by oralroute. The drug was administered in the food (Altromin, powdered food)and the dosage was calculated from the daily food intake. The symptomswere evaluated daily for each individual paw from 0.5-3 as described [R.Holmdahl, et al., Immunology, 65, 305-310, (1988)]. The total symptomsof every animal in each group—on day 7 after the booster injection—areshown in the following Table:

Treatment Total Symptoms Control 0 Collagen 35 Collagen/50 mg/kg C11AG4.5 Collagen/100 mg/kg C11AG 1

20 days after the booster injection the animals were killed and thejoints were examined by histopathology resulting in the followingpicture:

In all the untreated animals, inflammatory processes were found, but inthe animals and controls treated with 50 and 100 mg/kg of C11AG, no suchinflammatory processes could be detected.

The results obtained seven days after the booster injection aregraphically shown in FIG. 2.

Example 6

Inhibition of neutral SMase Compound Neutral SMase IC₅₀ [μM]Octylidene-aminoguanidine 63 Decylidene-aminoguanidine 44Undecylidene-aminoguanidine 8.2 Dodecylidene-aminoguanidine 5.8Anthracen-9-ylmethylene-aminoguanidine 1.9Indol-3-ylmethylene-aminoguanidine 5 Phenalen-1-ylidene-aminoguanidine54

¹⁴C sphingomyelin (10 μg/ml) was incubated with neutral SMase (membranefraction isolated from mice brains, 10 μg of protein/mixture [accordingto S. Gatt, Biochem. Biophys. Res. Commun. 68, 235-241 (1976)] in thepresence of various concentrations of the test substances (for 2 hoursat 37° C. 20 mM Tris, 1 mM MgCl₂.pH 7.5). Then the samples wereextracted with 5 times the volume of chloroform/methanol (1:1) and thecontent of radioactive phosphorylcholine in the aqueous phase wasdetermined. The IC₅₀ was obtained from dosage/activity curves.

Example 7

Inhibition of NO-synthase Induction by C11AG in Macrophages

RAW cells (mouse macrophage line, origin: American Type CultureCollection) were treated with 10 ng/ml of endotoxin from E. coli (LPS)in the presence of different concentrations of C11AG. After 16 hours thenitrite content in the culture medium was measured using the methoddescribed [K. Tschaikowsky, M. Meisner, F. Schonhuber and E. Rugheimer,Br. J. Pharmacol. 113 (3): 664-8 (1994)].

Measured Values:

C11AG concentration [μg/ml] OD 540 nm 0 0.122 0.5 0.091 1 0.075 2 0.0543 0.05 4 0.038

The inhibition of NO-synthase induction is graphically shown in FIG. 3;the NO₂ concentration [OD measured at 540 nm] is plotted against theC11AG concentration [μg/ml] for 10 ng/ml of LPS.

Example 8

C11AG-IC₅₀ determination of acidic and neutral SMase ¹⁴C sphingomyelin(10 μg/ml) were incubated with neutral SMase (membrane fraction isolatedfrom mouse brains, 10 μg protein/mixture, according to Gatt, S. Biochem.Biophys. Res. Commun. 68, 235-241, 1976) or with acidic SMase (microsomefraction from macrophage 5 μg of protein/mixture isolated according toGatt, S. Biochem. Biophys. Res. Commun. 68, 235-241, 1976) in thepresence of various concentrations of the test substances, for 2 hoursat 37° C. in 20 mM Tris, 1 mM MgCl₂, pH 7.5 (neutral SMase) or in 50 mMsodium acetate, 1 mM MgCl₂₁ pH 5.6 (acid SMase). Then the samples wereextracted with 5 times the volume of chloroform/methanol (1:1) and thecontent of radioactive phosphorylcholine in the aqueous phase wasdetermined. The release of phosphorylcholine in the untreated mixturescorresponds to 100% enzyme activity.

Measured Values:

C11AG concentration nSMase activity aSMase activity [μg/ml] [%] [%] 0100 100 1 61 101 10 18 102 100 0 31

FIG. 4 shows, by a simple logarithmic representation, thesphingomyelinase inhibition for neutral and acidic sphingomyelinase [in%] as a function of the C11AG concentration [μg/ml].

Example 9

Inhibition of the Growth of Papillomas

Mastomys natalensis with papillomas triggered by a papilloma virus [seeE. Amtmann and K. Wayss: The Mastomys natalensis papilloma virus, in: P.Salzman and P. Howley (Eds.). The Papovaviridae, Vol. 2. PlenumPublishing Corporation (1987)] were given food containing variousamounts of C11AG. The food consumption was measured and from this thedaily oral dose of C11AG was calculated. The size of the papilloma wasmeasured in two dimensions by means of a sliding gauge and the relativegrowth was calculated. 10 animals were treated per dose.

FIG. 5 graphically shows the average tumour size as a function of theduration of treatment for various doses of C11AG. Curve A shows thetumour growth of the control animals. Curve B shows the pattern of sizefor a dosage of 50 mg/kg C11AG and curve C shows the correspondingpattern for 100 mg/kg C11AG.

Example 10

Hydrogenated C11AG-IC₅₀: Measurement of Acidic and Neutral SMase

¹⁴C-sphingomyelin (10 μg/ml) was incubated with neutral SMase (membranefraction from mouse brain, 10 μg of protein/batch, isolated according toGatt, S. Biochem. Biophys. Res. Commun. 68, 235-241, 1976) or with acidSMase (microsome fraction from macrophages 5 μg of protein per batch[isolated according to S. Gatt, Biochem. Biophys. Res. Commun. 68,235-241, (1976)] in the presence of various concentrations of the testsubstances for 2 hours at 37° C. in 20 mM Tris, 1 mM MgCl₂, pH 7.5(neutral SMase) or in 50 mM sodium acetate, 1 mM MgCl₂, pH 5.6 (acidicSMase). Then the samples were extracted with 5 times the volume ofchloroform/methanol (1:1) and the content of radioactivephosphorylcholine in the aqueous phase was determined. The release ofphosphorylcholine in the untreated batches corresponds to 100%.

Measured Values:

H₂C11AG concentration nSMase activity aSMase activity [μg/ml] [%] [%] 0100 100 1 73 100 10 22 97 100 2 32

FIG. 6 shows, by a simple logarithmic representation, thesphingomyelinase inhibition for neutral—curve A—and acidicsphingomyelinase [in %]—curve B—as a function of the H₂C11AGconcentration [μg/ml].

Example 11

Prevention of Lethal Endotoxic Shock in the Mouse by H₂C₁₁AG

10 mice of the Balb C strain (about 8 weeks old) were given 0.7 mg ofendotoxin from E. coli (in 0.2 ml of isotonic saline solution) byintraperitoneal injection. 10 animals were given 100 mg/kg of H₂C11AG(dissolved in twice distilled water) by oesphageal tube 2 hours beforethe LPS treatment. The control animals were given water. The survivinganimals were observed for 12 days.

Results: control: 2 survivors (20%), 100 mg/kg H₂C11AG (hydrogenatedC11AG): 9 survivors (90%).

FIG. 7 shows the survival rate of untreated experimental animals (A)compared with those who were treated with a dose of 100 mg H₂C11AG, asdescribed above.

To illustrate the nomenclature used in the application, here are thestructures of some of the compounds mentioned:

1-(Anthracen-9-ylmethylene-amino)guanidine

1-(Indol-3-ylmethylene-amino)guanidine

1-(Phenalen-1-ylidene-amino)guanidine

What is claimed is:
 1. A compound having the formula:

wherein X denotes —NH—NH—CH₂R₁ and R₁ denotes C₈ to C₂₀ -alkyl, eitherbranched or unbranched; and optionally in the form of the individualoptical isomers, mixtures of the individual isomers or racemates,tautomers and the corresponding acid addition salts withpharmaceutically acceptable acids.
 2. A pharmaceutical compositioncomprising a compound of claim 1 with pharmaceutically acceptable acidstogether with pharmaceutically acceptable excipients and carriers.
 3. Acompound according to claim 1, wherein R₁ denotes an unbranched decylgroup.
 4. A compound according to claim 1 being1-(undecylamino)guanidine.
 5. A process for preparing a compound of theformula

, wherein R₁ represents C₈ to C₂₀ alkyl, either branched or unbranched,said process comprising a first step of reacting an aldehyde having thegeneral formula R₁CHO with an aminoguanidine, a second step of reducingthe imine function resulting from the first step in the presence of ahydrogenation catalyst under elevated hydrogen pressure and isolatingthe reaction product, and optionally forming the corresponding acidaddition salt with a pharmaceutically acceptable acid.
 6. The compoundof claim 1, in the form of a racemic mixture.